We thank Ms. J. Shinohara and I. Nieda for technical and clerical work.&#160;Also, we thank Drs. Shiro Matsumoto, Hidenori Haruta, Kentaro Kurashina, and Kazuya Takahashi for their cooperation for sample acquisition in operating room.&#160;This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan and the Japan Society for the Promotion of Science (17K10606).

LDN were obtained from patients enrolled in this study and were approved by the Institutional Review Board of Jichi Medical University.
1. Isolation of LDN from Abdominal Cavity Lavages and NET Detection
<ol>
	<li>Sample acquisition
	<ol>
		<li>Infuse 1,000 mL of normal sterile saline directly into the abdominal cavity before wound closure in patients who have undergone abdominal surgery due to gastrointestinal malignancy. 
		NOTE: Samples were obtained from patients who underwent a gas...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-density+granulocytes:+a+distinct+class+of+neutrophils+in+systemic+autoimmunity.">Low-density granulocytes: a distinct class of neutrophils in systemic autoimmunity.</a> Seminars in Immunopathology. 35 (4), 455-463 (2013).</li><li>Villanueva, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Netting+neutrophils+induce+endothelial+damage,+infiltrate+tissues,+and+expose+immunostimulatory+molecules+in+systemic+lupus+erythematosus.">Netting neutrophils induce endothelial damage, infiltrate tissues, and expose immunostimulatory molecules in systemic lupus erythematosus.</a> The Journal of Immunology. 187 (1), 538-552 (2011).</li><li>Brinkmann, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps+kill+bacteria.">Neutrophil extracellular traps kill bacteria.</a> Science. 303 (5663), 1532-1535 (2004).</li><li>Demers, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancers+predispose+neutrophils+to+release+extracellular+DNA+traps+that+contribute+to+cancer-associated+thrombosis.">Cancers predispose neutrophils to release extracellular DNA traps that contribute to cancer-associated thrombosis.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (32), 13076-13081 (2012).</li><li>Cools-Lartigue, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps+sequester+circulating+tumor+cells+and+promote+metastasis.">Neutrophil extracellular traps sequester circulating tumor cells and promote metastasis.</a> Journal of Clinical Investigation. , (2013).</li><li>Tohme, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+Extracellular+Traps+Promote+the+Development+and+Progression+of+Liver+Metastases+after+Surgical+Stress.">Neutrophil Extracellular Traps Promote the Development and Progression of Liver Metastases after Surgical Stress.</a> Cancer Research. 76 (6), 1367-1380 (2016).</li><li>Monti, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin-dependent+cell+adhesion+to+neutrophil+extracellular+traps+through+engagement+of+fibronectin+in+neutrophil-like+cells.">Integrin-dependent cell adhesion to neutrophil extracellular traps through engagement of fibronectin in neutrophil-like cells.</a> Public Library of Science One. 12 (2), e0171362 (2017).</li><li>Najmeh, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps+sequester+circulating+tumor+cells+via+beta1-integrin+mediated+interactions.">Neutrophil extracellular traps sequester circulating tumor cells via beta1-integrin mediated interactions.</a> International Journal of Cancer. 140 (10), 2321-2330 (2017).</li><li>Najmeh, S., Cools-Lartigue, J., Giannias, B., Spicer, J., Ferri, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+Human+Neutrophil+Extracellular+Traps+(NETs)+Isolation+and+Handling.">Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling.</a> Journal of Visualized Experiments. (98), (2015).</li><li>Kanamaru, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+density+neutrophils+(LDN)+in+postoperative+abdominal+cavity+assist+the+peritoneal+recurrence+through+the+production+of+neutrophil+extracellular+traps+(NETs).">Low density neutrophils (LDN) in postoperative abdominal cavity assist the peritoneal recurrence through the production of neutrophil extracellular traps (NETs).</a> Scientific Reports. 8 (1), 632 (2018).</li><li>Eades-Perner, A. M., Thompson, J., van der Putten, H., Zimmermann, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mice+transgenic+for+the+human+CGM6+gene+express+its+product,+the+granulocyte+marker+CD66b,+exclusively+in+granulocytes.">Mice transgenic for the human CGM6 gene express its product, the granulocyte marker CD66b, exclusively in granulocytes.</a> Blood. 91 (2), 663-672 (1998).</li><li>Park, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+cells+induce+metastasis-supporting+neutrophil+extracellular+DNA+traps.">Cancer cells induce metastasis-supporting neutrophil extracellular DNA traps.</a> Science Translational Medicine. 8 (361), 361ra138 (2016).</li></ol>

The authors declare that they have no competing financial interests.

Previous studies have reported that circulating tumor cells can be trapped by NET substrates in vivo10,11. Metastatic breast cancer cells have been shown to stimulate neutrophils and induce formation of NETs, which assists in tumor cell growth in the target organ17. In addition, we found that short-term cultures of LDN from postoperative lavage fluid produced massive NETs that could efficiently entrap tumor cells without further s...

Polymorph nuclear neutrophils in circulating blood are typically separated from mononuclear cells through the density gradient preparation method. However, some neutrophils known as low-density neutrophils (LDN), with CD11b(+), CD15(+), CD16(+), and CD14(-) phenotypes, are co-purified with mononuclear cells. The relative number of LDN significantly increases in various pathological conditions including autoimmune diseases1,2, sepsis3, and cancer4,5. Previous studies have shown that LDN are a phenotypically and functionally distinct class of neutrophils6. It should be noted that LDN in circulating blood are more likely to produce neutrophil extracellular traps (NETs) than normal density neutrophils2,7. NETs are web-like structures composed of nucleic acids, histones, proteases, and granular and cytosolic proteins, and they can efficiently entrap and destroy pathogens8.
Recently, NETs have been shown to capture not only microbes, but also platelets and circulating tumor cells which can assist in thrombus formation9 and tumor metastases10,11. However, the molecular mechanisms behind the adhesive interactions between NETs and platelets or tumor cells are still unclear. More recently, an in vitro adhesion assay revealed that myeloid leukemia cells (K56212) and lung carcinoma cells (A54913) attach to NETs via &#946;1 and &#946;3 integrins. The authors used NET stock isolated from neutrophils and activated by phorbol 12-myristate 13-acetate (PMA) as the adhesion substrate14. Although this assay allows detection of real interactions with NET components in the absence of neutrophils, it is arguable whether the &#34;cell-free NET stock&#34; isolated by high-speed centrifugation retains the molecular structure identical to NETs produced in vivo. Recently, we found that peritoneal lavage fluid after abdominal surgery contained many mature LDN, which generated massive NETs and attached to tumor cells causing peritoneal metastases15. In this study, we successfully examined the adhesion of tumor cells to intact NETs without any physical manipulation. Here, we show details of a technique to detect adhesive interactions between NETs and free tumor cells.

<table>
	<tbody>
		<tr>
			<td>Ficoll-Paque PLUS</td>
			<td>GE Healthcare, SWEDEN</td>
			<td>17-1440-02</td>
			<td></td>
		</tr>
		<tr>
			<td>StraightFrom&#8482; Whole Blood CD66b MicroBeads</td>
			<td>Miltenyi Biotec, Bergisch Gladbach, Germany</td>
			<td>130-104-913</td>
			<td></td>
		</tr>
		<tr>
			<td>Fc block</td>
			<td>Miltenyi Biotec, Bergisch Gladbach, Germany</td>
			<td>130-059-901</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS Rinsing Solution</td>
			<td>Miltenyi Biotec, Bergisch Gladbach, Germany</td>
			<td>130-091-222</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS BSA Stock Solution</td>
			<td>Miltenyi Biotec, Bergisch Gladbach, Germany</td>
			<td>130-091-376</td>
			<td></td>
		</tr>
		<tr>
			<td>LS Columns</td>
			<td>Miltenyi Biotec, Bergisch Gladbach, Germany</td>
			<td>130-041-306</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS Magnetic Separator</td>
			<td>Miltenyi Biotec, Bergisch Gladbach, Germany</td>
			<td>130-042-501</td>
			<td></td>
		</tr>
		<tr>
			<td>SYTOX green nucleic acid stain 5mM solution in DMSO</td>
			<td>Thermo Fisher Scientific, Waltham, MA, USA</td>
			<td>S7020</td>
			<td></td>
		</tr>
		<tr>
			<td>PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling</td>
			<td>Sigma-Aldrich, St Louis, MO, USA</td>
			<td>P9691</td>
			<td></td>
		</tr>
		<tr>
			<td>Diluent C</td>
			<td>Sigma-Aldrich, St Louis, MO, USA</td>
			<td>CGLDIL</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI1640 Medium</td>
			<td>Sigma-Aldrich, St Louis, MO, USA</td>
			<td>R8758</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium-high glucose (DMEM)</td>
			<td>Sigma-Aldrich, St Louis, MO, USA</td>
			<td>D5796</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline (DPBS)</td>
			<td>Sigma-Aldrich, St Louis, MO, USA</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5mol/l-EDTA Solution (pH 8.0)</td>
			<td>nacalai tesque, Japan</td>
			<td>06894-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, qualified, USDA-approved regions</td>
			<td>gibco by life technologies, Mexico</td>
			<td>10437-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin lyophilized powder, &#8805;96% (agarose gel electrophoresis)</td>
			<td>Sigma-Aldrich, St Louis, MO, USA</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Life Technologies Japan</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmocin Prophylactic</td>
			<td>InvivoGen, San Diego, CA-USA</td>
			<td>ant-mpp</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Worthington, Lakewood NJ)</td>
			<td>LS002138</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Lysine-Coated MICROPLATE 6Well</td>
			<td>IWAKI, Japan</td>
			<td>4810-040</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Lysine-Coated MICROPLATE 24Well</td>
			<td>IWAKI, Japan</td>
			<td>4820-040</td>
			<td></td>
		</tr>
		<tr>
			<td>fluorescein stereomicroscope</td>
			<td>BX8000, Keyence, Osaka Japan</td>
			<td>BZ-X710</td>
			<td></td>
		</tr>
		<tr>
			<td>Whole view cell observation system</td>
			<td>Nikon, Kanagawa, Japan</td>
			<td>BioStudio (BS-M10)</td>
			<td></td>
		</tr>
		<tr>
			<td>MKN45 human gastric cancer cell line</td>
			<td>Riken, Tukuba Japan</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>NUGC-4 human gastric cancer cell line</td>
			<td>Riken, Tukuba Japan</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>OCUM-1 human gastric cancer cell line</td>
			<td>Osaka City University, Japan</td>
			<td>N/A</td>
			<td>Gift from Dr. M.Yashiro</td>
		</tr>
	</tbody>
</table>

neutrophil extracellular traps generated by low density neutrophils obtained from peritoneal lavage fluid mediate tumor cell growth and attachment

Activated neutrophils release neutrophil extracellular traps (NETs), which can capture and destroy microbes. Recent studies suggest that NETs are involved in various disease processes, such as autoimmune disease, thrombosis, and tumor metastases. Here, we show a detailed in vitro technique to detect NET activity during the trapping of free tumor cells, which grow after attachment to NETs. First, we collected low density neutrophils (LDN) from postoperative peritoneal lavage fluid from patients who underwent laparotomies. Short-term culturing of LDN resulted in massive NET formation that was visualized with green fluorescent nuclear and chromosome counterstain. After co-incubation of human gastric cancer cell lines MKN45, OCUM-1, and NUGC-4 with the NETs, many tumor cells were trapped by the NETs. Subsequently, the attachment was completely abrogated by the degradation of NETs with DNase I. Time-lapse video revealed that tumor cells trapped by the NETs did not die but instead grew vigorously in a continuous culture. These methods may be applied to the detection of adhesive interactions between NETs and various types of cells and materials.

In the 2-hour culture, CD66b(+) LDN derived from peritoneal lavage fluid showed string structures stained with green-fluorescent dye for nuclear and chromosome (Figure 1B), while CD66b(-) mononuclear cells did not (Figure 1C). However, when the LDN cultures were pretreated with 100 U/mL DNase I, the characteristic structure was destroyed (Figure 1D), indicating that they were composed of extracellula...

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Neutrophil Extracellular Traps Generated by Low Density Neutrophils Obtained from Peritoneal Lavage Fluid Mediate Tumor Cell Growth and Attachment

Here, we present a method in which human low-density neutrophils (LDN), recovered from postoperative peritoneal lavage fluid, produce massive neutrophil extracellular traps (NETs) and efficiently trap free tumor cells that subsequently grow.

Activated neutrophils release neutrophil extracellular traps (NETs), which can capture and destroy microbes. Recent studies suggest that NETs are involved ...

This method can help answer key questions about the role of neutrophil subpopulations in tumor immunology especially about the mechanical involvement of neutrophil extracellular traps, or NETs, in tumor metastasis. The main advantage of this technique is that it allows visualization of the adhesive interactions of tumor cells to intact NETs. The implications of this technique extend towards the therapy of cancer.As NETs derived from specific neutrofil subsets may promote tumor metastasis. Demonstrating the procedure will be Junko Shoniohara from our laboratory. To acquire the neutrophils first infuse one liter of normal sterile saline immediately before wound closure directly into the abdominal cavity of a patient who has just undergone abdominal surgery due to gastrointestinal malignancy and lavage the abdominal cavity extensively for at least one minute.Then stir the saline within the cavity and recover 200 milliliters of lavaged fluid with four 50-milliliter syringes. In the lab transfer the peritoneal lavage fluid through a 100 micrometer nylon filter into individual 50 milliliter tubes for centrifugation. Re-suspend the pellets in 5 milliliters of PBS supplemented with 0.02%EDTA and carefully overlay each cell suspension onto three milliliters of density gradient solution.After density gradient separation harvest two to three milliliters of the intermediate layer from each tube and wash the peritoneal fluid samples in 10 milliliters of fresh PBS plus EDTA per tube. Pool the pellets in 10 milliliters of fresh PBS plus EDTA for an additional wash and dilute the cells to a one times 10 to the seventh cells per 60 microliters of magnetic activated cell sorting, or MACS, buffer concentration. Block any nonspecific binding with 20 microliters of Fc block for 10 minutes at four degrees Celsius followed by the addition of 20 microliters of anti-CD66b magnetic beads.After 10 minutes at four degrees Celsius wash and re-suspend the cells in 500 microliters of MACS buffer and add the cells to an appropriately sized magnetic column within the magnetic field of a suitable magnetic separator. When all of the CD66b negative cells have run through the column add 15 millilitres of fresh MACS buffer to the column reservoir and transfer the column from the magnetic separator into a new conical tube. Then immediately plunge the column to flush the magnetically labeled CD66b positive into the tube.After centrifugation re-suspend the isolated low density neutrophils at a five times 10 to the sixth cells per milliliter of RPMI1640 medium supplemented with FBS concentration and seed 1 milliliter of neutrophils per well on the six well poly-L-lysine coated plate. After two hours at 37 degrees Celsius and 5%carbon dioxide stain the cells in each well with an appropriate fluorescent membrane impermeable nuclear and chromosomal dye according to the manufacturer's instructions and immediately observe the morphology of the NETs by fluorescence microscopy. To assess tumor cell adhesion to the NETs re-suspend gastric tumor cells stained with the contrasting fluorescent dye at a one times 10 to the sixth per milliliter of RPMI1640 supplemented with 0.1%bovine serum albumin concentration and add one milliliter of tumor cells to each well of the low density neutrophil cultures.After five minutes at 37 degrees Celsius remove the supernatant from each well and gently wash the cells two times with 2 milliliters of pre warmed RPMI1640 medium supplemented with BSA per wash. A five minute incubation is long enough for detecting NET specific binding. Add the warmed medium to the side of each well slowly gently swirl the plate and remove the supernatant with an aspirator.The NETs and the attached tumor cells can then be observed by fluorescence microscopy. For a time lapse video analysis of the trapped tumor cells culture and stain the peritoneal low density neutrophils in a 35 millimeter dish, poly-L-lysine coated dish, as demonstrated followed by a brief wash in two milliliters of 0.1%BSA plus RPMI1640. Replace the medium with one times 10 to the sixth unstained gastric tumor cells suspended in two milliliters of RPMI supplemented with BSA for a five minute incubation at room temperature.At the end of the incubation rinse the cells in two milliliters of fresh RPMI supplemented with BSA for five minutes at room temperature followed by gentle swirling to remove any unattached tumor cells. Then replace the supernatant with DMEM supplemented with FBS and antibiotics and mount the dish in a whole view cell observation system for time lapse analysis of tumor cell trapping by the NETs. After two hours of culture CD66b positive low density neutrophils derived from peritoneal lavage fluid exhibit string structures by green fluorescent nuclear and chromosomal staining while CD66b negative mononuclear cells do not.When the low density neutrophil cultures are pre-treated with 100 units per milliliter of DNAse I, however, the characteristics structures are destroyed indicating that the structures are composed of extracellular DNA expelled from the neutrophils. When the low density neutrophils are cultured on uncoated plastic plates many NET clusters can be observed floating within the medium with few NETs attached to the bottom of the dish. After five minutes of incubation with a fluorescently labeled gastric tumor cell line many tumor cells attach to the neutrophils within the NETs a phenomenon that is not observed after pre-treatment with DNAse I.Similar adhesion patterns are also seen for other tumor cells.OKUM-1 and NUGC-4. Interestingly while the NETs gradually disappear within several hours of extended tumor cell co-culture the tumor cells trapped by the NETs begin to proliferate vigorously after NET release. During this procedure it's important to remember that because the NETs stick to the plate weakly the washing and dish transfer processes should be performed in an especially cautious manner.After its development this technique paves the way for researchers in various fields of biomedical science to explore the causal relationship of NETs with various diseases such as thrombosis, systemic inflammatory response syndrome, and autoimmune diseases.

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8.6K Views.  Jichi Medical University. This method can help answer key questions about the role of neutrophil subpopulations in tumor immunology especially about the mechanical involvement of neutrophil extracellular traps, or NETs, in tumor metastasis. The main advantage of this technique is that it allows visualization of the adhesive interactions of tumor cells to intact NETs. The implications of this technique extend towards the therapy of cancer.As NETs derived from specific neutrofil subsets may promote tumor metastas...

Video: Neutrophil Extracellular Traps Generated by Low Density Neutrophils Obtained from Peritoneal Lavage Fluid Mediate Tumor Cell Growth and Attachment